Diagnostic compositions comprising a complex formed by a nitrogenous macrocyclic ligand with metal ions

ABSTRACT

The invention relates to ligands having the formula (I), in which X represents a heterocyclic group, together with the process for their preparation. The invention also concerns mono- or polymetallic complexes formed by said ligands with metallic ions selected from lanthanide ions, transition metal ions, barium ion, bismuth ion, lead ion and the radioisotopes 99m Tc , 111 In , 90 Y , 64 Cu  and 169 Yb , as well as salts physiologically acceptable to these complexes. The salts of said complexes can be used as diagnostic and therapeutic agents in magnetic resonance imaging, radiology, in vivo chemical displacement agents and in nuclear medicine.

This case was filed under 35 U.S.C. §371 and is the national stage ofPCT/FR90/00198, filed Mar. 24, 1989. This is a continuation ofapplication Ser. No. 08/237,914, filed May 4, 1994, now abandoned, whichin turn is a continuation of application Ser. No. 07/768,391, filed Nov.20, 1991, abandoned.

The present invention relates to diagnostic compositions comprising acomplex formed by a macrocyclic ligand with metal ions which can be usedin particular in magnetic resonance imaging, in radiology using X-rays,and as in vivo chemical shift agents.

Thus, the object of the invention is diagnostic compositions comprisinga neutral or anionic, mono- or polymetallic complex formed by a ligandof formula I ##STR1## in which A and B each independently represents alinear or branched C₁ -C₈ alkylene group, a linear or branched C₁ -C₈hydroxyalkylene group, a linear or branched C₁ -C₈ polyhydroxyalkylenegroup, a --(CH₂ --CH₂ --O)_(z) --CH₂ --CH₂ -- group, z being an integerfrom 1 to 3, or at least one of the groups A or B may be represented bythe group: ##STR2## p being equal to an integer from 1 to 7, q beingequal to an integer from 0 to 8, and

R₄ representing a group making possible the binding of the macrocycle offormula I to a biomolecule or to a polymer, or a group of formula:##STR3## in which one of the groups A', B' or D' denotes ##STR4## pbeing as previously defined, the other groups A', B' or D' representingA, B or D, respectively,

n is equal to 0 or 1,

D is selected from the groups A, B and a group X, X being selected fromthe groups ##STR5## in which R₃ is selected from hydrogen, a C₁ -C₅alkyl group, a C₁ -C₅ hydroxyalkyl group and a C₁ -C₅ polyhydroxyalkylgroup,

R₁ is selected from the groups --CH₂ --COOH and --CH₂ --PO₃ H₂, and thecorresponding anions --CH₂ COO⁻ and --CH₂ --PO₃ ⁻⁻,

R₂ is selected from hydrogen, a C₁ -C₄ alkyl group, a C₁ -C₄hydroxyalkyl group, a C₁ -C₄ polyhydroxyalkyl group, a group ##STR6## inwhich R₅ and R₆ are each independently selected from hydrogen, a C₁ -C₄alkyl group, a C₁ -C₄ hydroxyalkyl group and a C₁ -C₄ polyhydroxyalkylgroup, the group ##STR7## A, B, D, R₁ and p being as previously definedas well as the --CH₂ --COOH and --CH₂ --PO₃ H₂ groups and thecorresponding anions --CH₂ --COO⁻ and CH₂ PO₂ ⁻⁻, with metal ionsselected from the ions of the lanthanides, the transition metals,barium, bismuth, lead and the radioisotopes: ^(99m) Tc, ¹¹¹ In, ⁹⁰ Y, ⁶⁴Cu and ¹⁶⁹ Yb, or a salt of this complex with a pharmaceuticallyacceptable mineral or organic base or a basic amino acid.

A group of ligands forming the complexes is constituted by the compoundsof formula I in which:

A represents a linear or branched C₂ -C₅ alkylene group,

B represents a linear or branched C₂ -C₅ alkylene group,

n is equal to 0 or 1,

D is selected from a linear or branched C₂ -C₅ alkylene group and agroup X, X being selected from the groups: ##STR8## in which R₃ isselected from hydrogen and a C₁ -C₃ alkyl group,

R₁ is selected from the groups --CH₂ --COOH and --CH₂ --PO₃ H₂, and

R₂ is selected from hydrogen a C₁ -C₄ alkyl group, a C₂ -C₄ hydroxyalkylgroup and a group R₁ as previously defined.

Another group of ligands forming the complexes is constituted by thecompounds of formula I in which n is equal to O and R₃ is selected froma C₁ -C₅ alkyl group, a C₁ -C₅ hydroxyalkyl group and a C₁ -C₅polyhydroxyalkyl group.

Another group of ligands forming complexes is also constituted by thecompounds of formula I in which n is equal to 1 and D represents the Xgroup, X being selected from: ##STR9## R₄ is preferably selected fromthe groups --NH₂, --CO₂ H, ##STR10## X⁻, X⁻ representing a monovalentanion such as Cl⁻ or BF₄ ⁻, ##STR11##

The biomolecules may be proteins, for example albumin or monoclonalantibodies or antibody fragments.

The polymers are selected from peptides, for example, polylysine and thepolysaccharides, for example Dextran.

In particular, the ligands constituted by the compounds of formula I inwhich A and B are identical and selected from the groups ethylene andn-propylene, n is equal to 1, X and D are identical and selected fromthe following groups, are preferred ##STR12## R₃ being selected fromhydrogen and methyl, and R₁ and R₂ represent the --CH₂ CO₂ H group arepreferred.

Another group of preferred ligands is constituted by the compounds offormula I in which A and B are identical and selected from ethylene andn-propylene, n is equal to O, X is selected from the groups: ##STR13##R₃ being selected from hydrogen and methyl, R₁ represents the --CH₂ CO₂H group and R₂ represents hydrogen or --CH₂ --CO₂ H.

Ligands having the following formulae are among those preferred:##STR14##

The ligands of formula I may be prepared by reaction of one mole ofpolyamine of general formula II ##STR15## in which A, B and n are aspreviously defined and D is selected from the groups A, B and a groupX₁, X₁ being selected from the groups: ##STR16##

either with 4 moles of a compound of formula III

    Y--CH.sub.2 --CO.sub.2 H

in which Y is a labile group such as a chlorine, bromine or iodine atomor a mesyloxy or tosyloxy group, in the presence of sodium or potassiumhydroxide when R₁ and R₂ denote --CH₂ CO₂ H, or with 4 moles offormaldehyde in the presence of phosphonic acid when R₁ and R₂ denote--CH₂ PO₃ H₂,

or with 3 moles of a compound of formula III such as described above inthe presence of sodium or potassium hydroxide when R₁ represents --CH₂CO₂ H, or with 3 moles of formaldehyde or phosphonic acid when R₁represents --CH₂ PO₃ H₂, then optionally with one mole of a compound offormula IV

    Y--R.sub.2

in which Y is as previously defined and R₂ is selected from a C₁ -C₄alkyl group, a C₁ -C₄ hydroxyalkyl group, a C₁ -C₄ polyhydroxyalkylgroup, the group ##STR17## R₅ and R₆ being as previously defined, andcatalytic hydrogenation of the X₁ groups is optionally performed in thepresence of a suitable metal catalyst such as Pd/C, PtO₂, Rh/C, RuO₂under high hydrogen pressure in order to produce the compound of formulaI in which X represents a saturated heterocycle (pyrrolidine,piperidine, tetrahydrofuran and tetrahydrothiophene).

The cyclic polyamines of formula II may be prepared by

a) cyclization as described by S. M. Nelson, Pure and Appl. Chem., 52,2461-2476 (1980), by Fenton, Pure and Appl. Chem. 58, 1437-1444 (1986),by Khalil, K. Abid et al. in Inorganica Chemica Acta, 82 (1984),223-226, by K. F. Dancey et al. in Synthetic Communications, 16 (7),795-801 (1986), by V. Mc Kee et al. in J. Chem. Soc., Chem. Commun.1983, 1465-1467 and by V. Mc Kee et al. in J. Chem. Soc., Chem. Commun.,1985, 158-159, from dicarbonyl compounds of formula V ##STR18## X₂ beingselected from the groups ##STR19## and R₃ being as previously definedwith polyamines of formula VI

    NH.sub.2 --A--NH.sub.2

and VII

    NH.sub.2 --B--NH.sub.2

or with a polyamine of formula VIII

    NH.sub.2 --A--NH--B--NH.sub.2

A and B being as previously defined, and

b) reduction of the Schiff base thus obtained by means of a metallicreducing agent such as Na BH₄ in a suitable solvent such as methanol toproduce the ligands of formula II.

The cyclization reaction takes place optionally in the presence of ametal cation suitable for "cyclization assisted by a metal", the metalbeing selected from the transition metals, the lanthanides, barium,calcium and strontium in which case the metal cation is removed aftercyclization by means of a compound selected from HBr, KCN and H₂ S.

The compounds of formula II may also be prepared as described in"Darstellung und Komplexbildung von Polyazacycloalkan essig-saure", H.Stetter et al., Tetrahedron, vol 37, pp. 767 to 772 , 1981 bycyclization of a compound of formula IX ##STR20## Y being a labile groupselected from chlorine, bromine, iodine, the tosyloxy or mesyloxy groupobtained from the corresponding alcohols with a polysulfonamide offormula X: ##STR21## in which Ts is a tosyl group and A, D, B and n areas previously defined, in the presence of a suitable base selected fromNaH, MeONa, EtONa and Cs₂ CO₃ in a suitable solvent such asdimethylformamide and dimethylacetamide or other aprotic solvents or inthe presence of a phase transfer catalyst under the conditions of phasetransfer known to the prior art.

The complexes are neutral or anionic mono- or polymetallic, andpreferably mono- or bi-metallic.

In these complexes, the metal ions are preferably gadolinium, europium,dysprosium, iron (Fe³⁺), manganese (Mn²⁺) and barium.

A preferred bimetallic complex is that formed with a ligand of formula Iin which X and D represent the group: ##STR22## A and B represent the2-hydroxy propylene group n=1 and R₁ and R₂ represent --CH₂ --COO⁻ withtwo Mn²⁺ ions.

Examples of salts are those formed with sodium hydroxide,N-methylglucamine, diethanolamine, lysine and arginine.

The complexes may be obtained by reaction of the ligands with a salt oran oxide of the metals in an aqueous solvent and neutralization, ifnecessary, to form a salt.

It is obvious that the present invention encompasses not only the use ofthe previously defined complexes in the form of racemic mixtures butalso the use of the stereoisomers of these complexes.

The complexes formed by the ligands of formula I may be used for invitro and in vivo diagnostic purposes in man and animals.

In magnetic resonance imaging and in in vivo NMR spectroscopy they maybe used as relaxation agents; for this purpose the complexes formed withthe following metals are preferred: Gd³⁺, Mn²⁺ and Fe³⁺ ; as magneticsusceptibility agents: for this purpose the complexes formed with themetals Dy³⁺, Ho³⁺, Tb³⁺ and Er³⁺ are preferred; or chemical shiftagents: for this purpose, the complexes formed with the metals Eu³⁺,Pr³⁺ and Yb³⁺ are preferred.

The complexes formed by the ligands of formula I and the metals selectedpreferably from Gd³⁺, Er³⁺, Dy³⁺, Tb³⁺, Ce³⁺, La³⁺, Bi²⁺, Ba²⁺ and Pb²⁺may be used in X-ray imaging.

In nuclear medicine, the complexes formed by the ligands of formula Iand the metals ^(99m) Tc, ¹¹¹ In, ⁶⁴ Cu and ¹⁶⁹ Yb may be used forradiodiagnostic purposes and the complexes formed with the metals ⁹⁰ y,²¹² Bi and ⁶⁴ Cu may be used for radiotherapeutic purposes aftercoupling of the complex to a suitable biomolecule.

The complexes formed by the ligands of formula I and the metal ionsselected preferably from Eu³⁺ and Tb³⁺ may be used in in vitrodiagnostic applications using photoluminescence, such asimmunofluorescence assays.

The compositions according to the invention may be constituted inparticular by solutions of a complex according to the invention in aphysiologically acceptable aqueous solvent.

The diagnostic compositions according to the invention may beadministered:

by the parenteral route, including the intravenous route, theintra-arterial route, the intra-lymphatic route and the subcutaneousroute

by the oral route

by the sub-arachnoidal route

by the intrabronchial route in the form of an aerosol.

In magnetic resonance imaging, the doses are very variable depending onthe route of administration.

In the case of the intravenous or intra-arterial route, the dose isabout 0.01 to 2 mM/kg.

In the case of the oral route, this dose may range up to 10 mM/kg.

For the other routes of administration, the useful doses are usuallylower than 1 mM/kg and even for the sub-arachnoidal route are usuallyless than 0.05 mM/kg.

The doses are the same for their use as chemical shift agents for invivo spectroscopy, as magnetic susceptibility agents in MRI and ascontrast agents in radiology using X-rays, except by the intravenous orintra-arterial route in which the doses may be from 0.2 to 5 mM/kg.

The following examples illustrate the preparation of the compoundsaccording to the present application.

In these examples:

the NMR spectra were performed on a Varian EM 360 apparatus at 60 MHzwith TMS as internal standard.

EXAMPLE 1

Preparation of the compound of formula: ##STR23##

A solution of 1,2-diaminoethane (0.03 mole) in 20 cm³ of methanol isadded dropwise to a solution of diformyl pyridine (4.05 g, 0.03 mole)and BaCl₂.2H₂ O (3.66 g, 0.015 mole) in 150 cm³ of methanol and thereaction mixture is heated at reflux for 3 hours.

After the solution has been cooled to 0° C., a first portion of NaBH₄(0.08 mole) is added slowly, a second addition of NaBH₄ (0.04 mole) ismade 30 minutes later.

The reaction mixture is stirred for 1 hour 30 at room temperature,concentrated to dryness and extracted with CHCl₃.

The oil obtained after evaporation is recrystallized from methanol inthe form of the tetrahydrobromide.

Yield=7.8 g (80%)

NMR in D₂ O: 7.34 ppm (triplet, J=8 Hz, 6.87 ppm (doublet, J=8 Hz), 3.95ppm (singlet), 3.24 ppm (singlet).

EXAMPLE 2

Preparation of the compound of formula: ##STR24##

A solution of sodium hydroxide (1.17 g in 3 cm³ of H₂ O) is added to asuspension of 1 (4.77 g, 0.007 mole) in 10 cm³ of water while thetemperature is maintained at 25° C.

15.28 g of bromoacetic acid, 9.53 g of 1 and a solution of sodiumhydroxide (6.75 g in 17 cm³ of H₂ O) are then added alternately in smallportions during a period of one hour. The temperature is maintainedbelow 60° C. After the addition, the reaction medium is adjusted topH=8.5 by the addition of sodium hydroxide (4.5 g in 10 cm³ of H₂ O) andmaintained at 45° C. for 36 hours.

The reaction mixture is then adjusted to pH=3 by the addition of HBr,then chromatographed on a resin (Dowex 50×4-400).

After the resin has been washed with water, the product is eluted with a0.5M solution of ammonia.

The fractions containing the product are adjusted to pH2 by the additionof concentrated HCl and evaporated to dryness. 15.4 g of product arerecovered in a yield of 94%.

¹ H NMR in D₂ O: 7.54 ppm (triplet J=8 Hz) 7.04 ppm (doublet J=8 Hz),4.02 ppm (singlet), 3.15 ppm (singlet), 2.98 ppm (singlet).

EXAMPLE 3

Preparation of the sodium salt of the Gd³ complex of the compound offormula 2. ##STR25##

The suspension of 0.3625 g of Gd₂ O₃ and 1.557 g of compound 2 in 10 cm³of H₂ O is heated at reflux for 4 hours. After being cooled to roomtemperature, the clear solution is neutralized by the addition of sodiumhydroxide to pH 7.2. On addition of acetone (150 cm³), 1.2 g of a whitesolid are recovered. The complex obtained is recrystallized bydissolution in a water/ethanol (40/60) mixture and addition of acetone.

Elementary analysis--calculated for NaGdC₂₆ H₃₁ N₆ O₈.5 : C 41.95; H4.17; N 11.29; Gd 21.14. Found: C 42.15; H 4.30; N 11.25; Gd 20.47.

EXAMPLE 4

Preparation of the compound of formula: ##STR26##

A solution of NaOH (60 mmoles, 2.6 g in 10 ml H₂ O) is added slowly withstirring to a suspension of the compound obtained in example No. 1 (15mmoles, 9.75 g) in water (30 ml) while the temperature is maintained atabout 40° C. by means of a water-bath.

The mixture is stirred while solid BrCH₂ COOH (15 mmoles, 2.085 g) isadded. The pH is maintained at about 8.5 by the continuous addition of aNaOH solution (90 mmoles, 3.6 g in 10 ml H₂ O) and the temperature ismaintained at 60° C. throughout the reaction.

After about 3 hours, 15 mmoles (2.09 g) of solid BrCH₂ COOH are againadded and after a further 4 hours the final quantity (15 mmoles, 2.09 g)of solid BrCH₂ COOH is added. During these additions, the pH ismaintained at 8.5 and the temperature at 60° C.

After 24 h, the pH of the reaction mixture is adjusted to 3.0 by theaddition of concentrated HBr.

The salts and excess reagents are removed from the product by cationexchange chromatography (Dowex 50×8).

The reaction mixture is loaded onto the column equilibrated and washedwith water until the eluate no longer gives a positive reaction tohalides and the pH is about 4.5.

The ligand is eluted from the column with 0.5 molar aqueous NH₃. Thefractions having a pH of 4.1 to 5.1 are pooled and evaporated to drynessunder reduced pressure (yield 6.0 g, 80% of crude product).

The solid is dissolved in 60 ml of CH₃ OH and added dropwise to astirred volume of acetone (500 ml) in order to give a very fine whitesolid.

The solid is recovered and dried in a vacuum at room temperature.

EXAMPLE 5

Preparation of the complex of formula: ##STR27##

Gd₂ O₃ (2.88 g, 8 mmoles) and the crude compound obtained in theprevious example (evaporated eluate obtained from the ion exchangecolumn, 7.96 g (16 mmoles) are suspended in 100 ml of H₂ O at 80° C. andstirred for 24 hours. During this time the solution becomes clear.

The solution is cooled to room temperature, then the solvent is removedunder reduced pressure.

The resultant solid is dissolved in 15 ml of H₂ O and 5 ml of C₂ H₅ OHand the solution is added dropwise to 750 ml of vigorously stirredacetone.

The fine, whitish precipitate which is formed is filtered off, thenwashed with acetone and dried in a vacuum at room temperature.

Yield: 6.8 g (66%).

EXAMPLE 6

Preparation of the compound of formula: ##STR28##

A solution of 1.2-diaminoethane (0.270 ml, 4 mmoles in 40 ml MeOH) isadded dropwise to a solution of 2,5-furandicarbaldehyde (0.505 g, 4mmoles) and Ba(SCN)₂.3H₂ O (0.615 g, 2 mmoles) in 60 ml MeOH during 15minutes.

The mixture is stirred for 3 hours and the colour turns orange-yellow.

The mixture is then cooled in an ice-bath while NaBH₄ (0.404 g, 10.7mmoles) is added slowly.

The colour of the reaction turns lemon yellow.

After stirring has been continued for 30 minutes, a further addition ofNaBH₄ (0.2018 g, 5.35 mmoles) is made.

After being stirred for 2 hours at room temperature, the mixture isevaporated to dryness under reduced pressure and the residue (a mixtureof white and yellow solids) is extracted five times with 25 ml aliquotsof CHCl₃.

The pooled chloroform extracts are filtered and evaporated under reducedpressure to give a yellow oil.

The oil is dissolved in MeOH (10 to 15 ml) and HBr (6 to 7 drops of a48% aqueous solution) is added dropwise to this solution. The productprecipitates in the form of a pale yellow solid.

The product is recovered, then dried in a vacuum at room temperature.

Yield: 0.6 g or 42%

Elementary analysis for C₁₆ H₃₁ N₄ O₃.5 Br₄ : calculated: C, 29.33; H4.77; N, 8.55; Br, 48.79. Found: C, 29.24; H, 4.81; N, 8.56; Br, 48.00.

EXAMPLE 7

Preparation of the compound of formula: ##STR29##

A solution of NaOH (1.6 g, 0.04 mole in 20 ml of H₂ O) is added slowlywith stirring to a suspension of the compound obtained in the previousexample (0.01 mole, 6.28 g) in water (30 ml) while the temperature ismaintained at about 30° by means of a water-bath.

The mixture is stirred while solid BrCH₂ COOH (0.02 mole, 2.78 g) isadded. The pH is maintained at about 8.5 by the continuous addition of aNaOH solution (3.2 g in 10 ml H₂ O) and the temperature is maintained at60° C. throughout the reaction.

After about 3.5 hours, a further addition of about 0.010 mole (1.39 g)of solid BrCH₂ COOH is added and after a further 3.5 hours, the finalquantity (0.010 mole, 1.39 g) of solid BrCH₂ COOH is added. During theseadditions, the pH was 8.5 and the temperature was maintained at 60° C.

After 24 hours, there is no further uptake of the NaOH solution and thereaction is stopped. The pH of the reaction mixture is adjusted to 2.7by addition of concentrated HBr.

The salts and excess reagents are removed from the product by cationexchange chromatography (Dowex 50×8).

The reaction mixture is loaded onto the column equilibrated and washedwith water until the eluate no longer gives a positive reaction tohalide and the pH is about 4.5.

The ligand is eluted from the column with 0.5M aqueous NH₃. Thefractions having a pH equal to 3.2 are pooled and evaporated to drynessunder reduced pressure (yield 2.7 g).

The solid is dissolved in 15 ml of CH₃ OH and added dropwise to 200 mlof vigorous stirred acetone to give a very fine white solid.

The solid is recovered and dried in a vacuum at room temperature.

Yield: 1,8 g, 50%.

Elementary analyis for C₂₄ H₃₃ N₄ O₁₀.5 ; Calc. C, 52.84; H, 6.10; N,10.27. Found: C, 53.06; H, 6.18; N, 9.67.

EXAMPLE 8

Preparation of the complex of formula: ##STR30##

Gd₂ (CO₃)₂ (0.3 g, 0.6 mmole) and the compound obtained in the previousexample (0.64 g, 1.2 mmole) are suspended in 40 ml of H₂ O at roomtemperature and stirred for 48 hours.

The suspension is filtered to give a colourless solution which isneutralized to pH 7 with a NaOH solution (0.05 g in 5 ml of H₂ O). Thesolvent is removed under reduced pressure.

The resultant solid is dissolved in 5 ml of H₂ O and 5 ml of C₂ H₅ OHand the solution is added dropwise to 200 ml of vigorously stirredacetone.

The fine white precipitate which is formed is recovered by filtration ata water pump, washed with acetone and dried in a vacuum at roomtemperature.

Yield: 0.56 g (65%).

EXAMPLE 9

Preparation of the compound of formula: ##STR31##

A solution of 1,3-diaminopropane (0.340 ml, 4 mmoles in 40 ml MeOH) isadded dropwise and with stirring to a solution of2,5-furandicarbaldehyde (0.505 g, 4 mmoles) and Ba(SCN)₂.3H₂ O (0.615 g,2 mmoles) in 60 ml of MeOH during 15 mn.

The mixture is stirred for 20 mn and the colour turns orange-yellow.

The mixture is then cooled in an icebath while NaBH₄ (0.404 g, 10.7mmoles) is added slowly.

The colour of the reaction turns lemon yellow.

After stirring has been continued for 30 mn, a further addition of NaBH₄(0.2018 g, 5.35 mmoles) is made.

After being stirred for 2 hours at room temperature, the mixture isevaporated to dryness under reduced pressure and the residue (a mixtureof white and yellow solids) is extracted four times with 15 ml aliquotsof CHCl₃.

The pooled chloroform extracts are filtered and evaporated under reducedpressure to give an orange oil.

The oil is dissolved in methanol (10 to 15 ml) and HBr (6 to 7 drops ofa 48% aqueous solution) is added dropwise to this solution. The productprecipitates in the form of a pale yellow solid.

The product is filtered off and dried in a vacuum at room temperature.

Yield: 0.67 g, 50%.

Elementary analysis: calculated for C₁₈ H₃₂ N₄ O₂ Br₄ : C, 32.95; H,4.92; N, 8.54; Br, 48.74. Found: C, 33.04; H, 4.96; N, 8.36; Br, 48.28.

EXAMPLE 10

Preparation of the compound of formula: ##STR32##

This compound is prepared according to the procedure described in theexamples 2 and 7.

EXAMPLE 11

Preparation of the compound of formula: ##STR33##

A solution of 1,2-diaminoethane (0.48 g, 8 mmoles in 20 ml MeOH) isadded dropwise and with stirring to a solution of 2,5-pyrroledicarbaldehyde (1.00 g, 8 mmoles) and BaCl₂.2H₂ O (0.976 g, 4 mmoles) in150 ml of methanol during 15 mn.

The initially yellow solution turns orange during the addition of theethylenediamine. The mixture is heated at reflux for 3 hours and thecolour turns dark brown.

The mixture is then cooled in an icebath while NaBH₄ (0.805 g, 21.3mmoles) is added slowly. The reaction becomes light brown on forming awhite precipitate.

After stirring has been maintained for 30 mn, a further addition ofNaBH₄ (0.404 g, 10.7 mmoles) is made.

After being stirred for 1.5 hour at room temperature, the mixture isevaporated to dryness under reduced pressure and the residue (a mixtureof an orange oil and a white solid) is extracted four times with 75 mlaliquots of CHCl₃.

The pooled chloroform extracts are filtered and evaporated under reducedpressure to give an orange oil.

The oil is dried in a vacuum at room temperature to give a light brownsolid.

Yield: 49%.

¹ H NMR in CDCl₃ : 10.08 ppm (large singlet, 2H, pyrrole NH), 5.95 ppm(singlet, 4H, pyrrole CH), 3.80 ppm (singlet, 8H, CH₂), 2.70 ppm(singlet, 8H, CH₂).

EXAMPLE 12

Preparation of the compound of formula: ##STR34##

This compound is prepared according to the procedure described in theexamples 2 and 7.

EXAMPLE 13

Preparation of the complex of formula: ##STR35##

This complex is prepared according to the procedure described in theforegoing examples 3 and 5.

EXAMPLE 14

Preparation of the compound of formula: ##STR36##

A solution of iminobis (propylamine) (1.4 ml, 0.01 mole in 10 ml C₂ H₅OH) is added dropwise and with stirring to a solution of2,6-pyridinedicarbaldehyde (1.35 g, 0.01 mole) and zinc triflate (3.63g, 0.01 mole) in 25 ml of C₂ H₅ OH and 25 ml of H₂ O during 15 mn.

NaBr (5.5 g) is added to the reaction mixture. The mixture is stirredfor 4 hours during which the product precipitates. The product isrecovered by filtration at a waterpump and is dried in a vacuum at roomtemperature for 12 hours. The product obtained (1.03 g, 1.7 mmole) isdissolved in 50 ml of methanol while NaBH₄ (0.48 g, 13 mmoles) is addedslowly.

After stirring has been maintained for 40 mn, a further addition ofNaBH₄ (0.44 g, 12 mmoles) is made.

After being stirred for 2 hours at room temperature, the mixture isevaporated to dryness under reduced pressure and the residue (a mixtureof whitish solids) is extracted four times with 25 ml aliquots of CHCl₃.The pooled chloroform extracts are filtered and evaporated under reducedpressure to give a cream coloured solid.

The solid is dissolved in methanol (60 ml) and HBr (about 3 ml of a 48%aqueous solution) is added dropwise to the stirred solution toprecipitate the product from the acidic medium as a pale yellow solid.

The product is recovered by filtration at a waterpump and is dried in avacuum at room temperature.

Yield: 0.52 g, 63%.

¹ H NMR in D₂ O: 7.93 ppm (triplet, area 1), 7.50 ppm (doublet, area 2),4.47 ppm (singlet, area 4), 3.05-3.35 ppm (multiplet, area 8), 2.25 ppm(multiplet, area 4).

EXAMPLE 15

Preparation of the compound of formula: ##STR37##

This compound is prepared by reaction with bromoacetic acid in thepresence of NaOH according to the procedure described in example 5above.

Similarly, the gadolinium complexes of the ligands corresponding to theexamples 10 and 15 are prepared in the presence of a stoichiometricamount of Gd₂ O₃ in aqueous medium according to the procedure describedin example 8.

What is claimed is:
 1. Method for in vivo imaging by nuclear magneticresonance or X-rays, comprising the steps of administering to a human oran animal an effective amount of a diagnostic composition and subjectingthe animal or human to a magnetic field or X-rays; said diagnosticcomposition comprising an effective amount of a neutral or anionic,mono- or polymetallic complex formed by:1) a ligand of formula ##STR38##in which: a) A and B each represents independently a linear or branchedC₁ -C₈ alkylene group, a linear or branched C₁ -C₈ hydroxyalkylenegroup, a linear or branched C₁ -C₈ polyhydroxyalkylene group, a --(CH₂--CH₂ --O)_(z) --CH₂ --CH₂ -- group, z being an integer from 1 to 3, ora group of formula ##STR39## i) p being equal to an integer from 1 to 7,ii) q being equal to an integer from 0 to 8, andiii) R₄ representingaa)a group for the binding of the macrocycle of formula I to a biomoleculeor a polymer, or bb) a group of formula ##STR40## in which one of A',B', and D' denotes ##STR41## p being previously defined, and two of A',B', and D' are two of A, B and D, respectively, b) n is 1, c) D is X, d)X is selected from the group consisting of ##STR42## in which R₃ isselected from the group consisting of hydrogen, C₁ -C₅ alkyl, C₁ -C₅hydroxyalkyl and C₁ -C₅ polyhydroxyalkyl, e) R₁ is selected from thegroup consisting of --CH₂ --COOH and --CH₂ --PO₃ H₂ and thecorresponding anions --CH₂ --COOH and --CH₂ --PO₃ ⁻⁻, f) R₂ is selectedfrom the group consisting of hydrogen, C₁ -C₄ alkyl, C₁ -C₄hydroxyalkyl, C₁ -C₄ polyhydroxyalkyl, --CH₂ --COOH, --CH₂ --PO₃ H₂,--CH₂ COO⁻, --CH₂ PO₃ ⁻⁻, a group ##STR43## in which R₅ and R₆ is eachindependently selected from the group consisting of hydrogen, C₁ -C₄alkyl, C₁ -C₄ hydroxyalkyl, and C₁ -C₄ polyhydroxyalkyl, and a group##STR44## X, A, B, D, R₁, n and p being as previously defined; and 2) ametal ion selected from the group consisting of Gd³⁺, Mn²⁺, Fe³⁺, Dy³⁺,Ho³⁺, Tb³⁺, Er³⁺, En³⁺, Pr³⁺, Yb³⁺, Pb³⁺, Ce³⁺, La³⁺, Bi²⁺, and Ba²⁺ ;ora salt of said complex with a pharmaceutically acceptable mineral ororganic base or a basic amino acid.
 2. Method for in vivo imaging bynuclear magnetic resonance or X-rays, comprising the steps ofadministering to a human or an animal an effective amount of adiagnostic composition and subjecting the animal or human to a magneticfield or X-rays; said diagnostic composition comprising an effectiveamount of a neutral or anionic, mono- or polymetallic complex formedby:1) a ligand of formula: ##STR45## in which: a) A represents a linearor branched C₂ -C₅ alkylene group,b) B represents a linear or branchedC₂ -C₅ alkylene group, c) n is 1, d) D is X, e) X is selected from thegroup consisting of ##STR46## in which R₃ is selected from the groupconsisting of hydrogen and a C₁ -C₃ alkyl group, f) R₁ is selected fromthe group consisting of --CH₂ --COOH and --CH₂ --PO₃ H₂, and g) R₂ isselected from the group consisting of hydrogen, C₁ -C₄ alkyl, C₂ -C₄hydroxyalkyl, --CH₂ --COOH, and --CH₂ CO₃ H₂ ; and 2) a metal ionselected from the group consisting of Gd³⁺, Mn²⁺, Fe³⁺, Dy³⁺, Ho³⁺,Tb³⁺, Er³⁺, En³⁺, Pr³⁺, Yb³⁺, Pb³⁺, Ce³⁺, La³⁺, Bi²⁺, and Ba²⁺ ;or asalt of said complex with a pharmaceutically acceptable mineral ororganic base or a basic amino acid.
 3. Method according to claim 1,wherein in the ligand of formula I R₄ is selected from the groupconsisting of --NH₂, --CO₂ H, ##STR47## X⁻ representing a monovalentanion Cl⁻ or BF₄ ⁻, ##STR48##
 4. Method according to claim 2, wherein inthe ligand of formula I R₄ is selected from the group consisting of--NH₂, --CO₂ H, ##STR49## X⁻ representing a monovalent anion Cl⁻ or BF₄⁻, ##STR50##
 5. Method according to claim 1, wherein in the ligand offormula I A and B are identical and selected from the group consistingof ethylene and n-propylene, n is equal to 1, X and D are identical andselected from the group consisting of: ##STR51## R₃ being selected fromthe group consisting of hydrogen and methyl, and R₁ and R₂ represent--CH₂ --CO₂ H.
 6. Method according to claim 2, wherein in the ligand offormula I A and B are identical and selected from the group consistingof ethylene and n-propylene, n is equal to 1, X and D are identical andselected from the group consisting of: ##STR52## R₃ being selected fromthe group consisting of hydrogen and methyl, and R₁ and R₂ represent--CH₂ --CO₂ H.
 7. Method according to claim 1, wherein in the ligand offormula I A and B represent a --CH₂ --CH₂ -- group, X and D representthe group: ##STR53## and R₁ and R₂ represent the --CH₂ --COOH group. 8.Method according to claim 2, wherein in the ligand of formula I A and Brepresent a --CH₂ --CH₂ -- group, X and D represent the group: ##STR54##and R₁ and R₂ represent the --CH₂ --COOH group.
 9. Method according toclaim 1, wherein in the ligand of formula I A and B represent a --CH₂--CH₂ -- group, X and D represent the group: ##STR55## R₁ represents the--CH₂ --COOH group and R₂ represents hydrogen.
 10. Method according toclaim 2, wherein in the ligand of formula I A and B represent a --CH₂--CH₂ -- group, X and D represent the group: ##STR56## R₁ represents the--CH₂ --COOH group and R₂ represents hydrogen.
 11. Method according toclaim 1, wherein in the ligand of formula I A and B represent the --CH₂--CH₂ -- group, X and D represent the group: ##STR57## and R₁ and R₂represent the --CH₂ --COOH group.
 12. Method according to claim 2,wherein in the ligand of formula I A and B represent the --CH₂ --CH₂ --group, X and D represent the group: ##STR58## and R₁ and R₂ representthe --CH₂ --COOH group.
 13. Method according to claim 1, wherein in theligand of formula I A and B represent the --(CH₂)₃ -- group, X and Drepresent the group: ##STR59## and R₁ and R₂ represent the --CH₂ --COOHgroup.
 14. Method according to claim 2, wherein in the ligand of formulaI A and B represent the --(CH₂)₃ -- group, X and D represent the group:##STR60## and R₁ and R₂ represent the --CH₂ --COOH group.
 15. Methodaccording to claim 1, wherein in the ligand of formula I A and Brepresent the --CH₂ --CH₂ -- group, X and D represent the group:##STR61## and R₁ and R₂ represent the --CH₂ --COOH group.
 16. Methodaccording to claim 2, wherein in the ligand of formula I A and Brepresent the --CH₂ --CH₂ -- group, X and D represent the group:##STR62## and R₁ and R₂ represent the --CH₂ --COOH group.
 17. Methodaccording to claim 1, wherein the composition comprises a mono- andbimetallic complex in which the metal ion is selected from the groupconsisting of gadolinium, europium, dysprosium, iron (Fe³⁺), manganese(Mn²⁺) and barium.
 18. Method according to claim 2, wherein thecomposition comprises a mono- and bimetallic complex in which the metalion is selected from the group consisting of gadolinium, europium,dysprosium, iron (Fe³⁺), manganese (Mn²⁺) and barium.
 19. Methodaccording to claim 1, wherein the composition comprises a bimetalliccomplex formed with a ligand of formula I in which X and D represent thegroup: ##STR63## A and B represent the 2-hydroxy propylene group, and R₁and R₂ represent --CH₂ --COO⁻ with two Mn²⁺ ions.
 20. Method accordingto claim 2, wherein the composition comprises a bimetallic complexformed with a ligand of formula I in which X and D represent the group:##STR64## A and B represent the 2-hydroxy propylene group, and R₁ and R₂represent --CH₂ --COO⁻ with two Mn²⁺ ions.
 21. Method according to claim1, in which the complex is coupled to a biomolecule or a polymer. 22.Method according to claim 2, in which the complex is coupled to abiomolecule or a polymer.
 23. Method according to claim 1, in which thecomposition is constituted by a solution of the complex in an aqueoussolvent.
 24. Method according to claim 2, in which the composition isconstituted by a solution of the complex in an aqueous solvent. 25.Method according to claim 1, wherein the metal ion is selected from thegroup consisting of Gd³⁺, Mn²⁺ and Fe³⁺.
 26. Method according to claim2, wherein the metal is selected from the group consisting of Gd³⁺, Mn²⁺and Fe³⁺.
 27. Method according to claim 1, wherein the metal is selectedfrom the group consisting of Gd³⁺, Er³⁺, Dy³⁺, Tb³⁺, Ce³⁺, La³⁺, Br²⁺,Ba²⁺ and Pb²⁺.